Real time quantification of target genes can provide best estimation of gene expression, although which relies mostly on efficient primers. The present study describes the design, synthesis and validation of novel primers targeting human toll like receptors (3, 7 and 9), involved in host innate immunity.Methodology: The primers were designed using ‘Beacon Designer software’, (Premier Biosoft, USA) and synthesized by IDT, USA. The primers were checked for specificity using NCBI-BLAST search and by visualization of specific sized product. Amplification efficiencies of primers were evaluated by generating standard curve in real time PCR (LC 480, Roche). Four sets of primers for commonly used housekeeping genes (ACTB, B2MG, GAPDH, 18srRNA) were tested for normalization of qPCR data.Results: The newly designed primers were specific, had comparable amplification efficiencies (1) and can be amplified at the same annealing temperature. ACTB and GAPDH were the most stable housekeeping genes. Relative quantification done in 20 each of hepatitis B positive cases and healthy individuals shown down regulation of TLR9 and no difference in expression with respect to TLR3 & TLR7 when using GAPDH as reference gene.Conclusion: The new sets of primers can be efficiently used for monitoring relative expression of genes targeting proteins for human toll like receptors- 3, 7 and 9.